Nanophotonic label-free biosensors for environmental monitoring

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On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.     

Genetically-encoded biosensors for monitoring cellular stress in bioprocessing

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Descriptions of new edaphic and aquatic species of Chlorococcum Meneghini (Chlorococcales)

The re-examination of plankton samples taken from Lough Neagh in 1900 by West & West (1902, 1906) and in 1910 by Dakin & Latarche (1913) indicates that Stephanodiscus astraea had been wrongly identified as Coscinodiscus lacustris. The absence of C. lacustris from the lake sediments is consequently explained.     

Enzyme inhibition-based biosensors for food safety and environmental monitoring

Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different contaminants analyzed are considered. The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.     

The development and applications of thermal biosensors for bioprocess monitoring

Enzyme thermistors are biosensors that use thermal resistors to measure the heat change caused by an enzymatic reaction. They combine the selectivity of enzymes with the sensitivity of biosensors and allow continuous analysis in a flow-injection mode. They can be used to monitor fermentation systems, biocatalysis, enzyme-catalysed synthesis and clinical and food technology. This article gives an overview of the general principles of enzyme thermistors, the sampling process and the ongoing developments in the field of bioprocess monitoring.     

Ecology of Vibrio mimicus in aquatic environments

An environmental study was done to examine the prevalence of Vibrio mimicus in some aquatic environments of Dhaka, Bangladesh, and of Okayama, Japan. Water samples from Dhaka environments and water and plankton samples from Okayama environments were quantitatively as well as qualitatively analyzed throughout the seasons for V. mimicus. The organism was isolated from Bangladesh environments throughout the year, whereas it was not isolated in Okayama when the water temperature fell below 10 degrees C. Samples with as many as 9.0 x 10(2) CFU of V. mimicus per 100 ml of water in Dhaka and 1.5 x 10(4) CFU of V. mimicus per 100 ml of water in Okayama were detected during the study period. V. mimicus was not found in any environment with an average salinity of 10% or more. Brackish environments with an average salinity of 4% were observed to be the optimal natural condition for the pathogen. Using the API 20E system with the conventional test methods, we observed variations in biochemical properties within the V. mimicus species. This study reveals the inefficacy of the API 20E system to identify a significant percentage of V. mimicus. Therefore, in addition to the API 20E system, a salt tolerance test and a string test are recommended for identification of this species. Susceptibility testing of strains isolated from Okayama environments showed higher resistance to ampicillin and susceptibility to trimethoprim-sulfamethoxazole when compared with environmental isolates of V. mimicus from Bangladesh.     

Significance of bacterial ectoenzymes in aquatic environments

The report presents studies on temporal and spatial variations of kinetics (Vmax and Km) of bacterial ectoenzyme activity (-glucosidase - Glc, leucine aminopeptidase - Leu-amp) in the naturally eutrophic Plusee. Glc and Leu-amp activity were positively correlated with the flux of polymeric materials (polysaccharides, proteins) in the lake. Glc activity was low when algal populations grew actively, but during the algal bloom breakdown Glc activity increased rapidly. Leu-amp displayed the highest rates of activity in the epilimnion and was tightly coupled to bacterial production. The synthesis of studied ectoenzymes was under control of a repression/derepression mechanism. The significance of ectoenzymes for the transformation and bacterial utilization of organic matter, and their role in the microbial loop in aquatic environments is discussed.     

Survival of enterococcal species in aquatic environments

Analysis of the survival ability of faecal streptococci/enterococci in the environment has almost invariably been conducted using the standard culture method (CFU counts) despite the demonstration that these microorganisms are capable of entering a viable but nonculturable (VBNC) state. In this study we evaluated the fate, in terms of culturability and viability, of different enterococcal species under laboratory stress conditions mimicking those of the aquatic environment. The results indicate that enterococcal species may activate two different survival strategies, namely starvation and the VBNC state, depending on the specific environmental condition. Moreover, the different enterococcal species can be divided into three groups on the basis of the time needed to activate the VBNC state and the resuscitation capability. The differences in activation of the two survival strategies and the different kinetics observed among the enterococcal species reaching the VBNC state should be taken into consideration when the microbiological quality of waters has to be evaluated and because of their role as faecal contamination indicators.     

Novel planar glucose biosensors for continuous monitoring use

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.     

Graphite-Teflon composite bienzyme amperometric biosensors for monitoring of alcohols

Composite graphite-Teflon electrodes, in which the enzymes alcohol oxidase (AOD) and horseradish peroxidase (HRP), as well as the mediator ferrocene, are incorporated into the electrode matrix, are reported for the reliable monitoring of alcohols in food and beverages. The bienzyme electrodes are constructed by simple physical inclusion of the enzymes and the mediator in the bulk of graphite-70% Teflon rigid cylindrical pellets. The composite biosensors are robust and reusable because of the renewability of the electrode surface by polishing. Reproducible amperometric responses at 0.00 V were obtained with different electrodes constructed from the same pellet and from different pellets. No significant loss of the enzymes activities was found after at least 3 months of storage at 0 degrees C. The detection limits obtained by amperometry in stirred solutions can be advantageously compared with those achieved with commercial sensors for alcohols. The bienzyme electrodes are suitable to be used under flow-injection conditions, as well as for amperometric detection in HPLC. The bioelectrodes were employed for the determination of ethanol in beers, wines and liquors, using both batch- and flow-injection modes, and for the determination of methanol in wines and liquors by HPLC with amperometric detection. Only a dilution of the beverages was needed as sample treatment in all cases.